Conformational Sensors and Domain Swapping Reveal Structural and Functional Differences between β-Arrestin Isoforms

Abstract

Highlights d b-arrestin 1 and 2 differentially regulate signaling and trafficking of GPCRs d There are conformational differences between receptorbound b-arrestin 1 and 2 d Domain-swapped chimera of b-arrestin 1 and 2 exhibit functional complementation d Functional differences of b-arrestin isoforms have implications for biased agonism

Key phosphorylation sites in GPCR s orchestrate the contribution of β‐Arrestin 1 in ERK 1/2 activation

“…For transfection, 50-60% confluent cells were transfected with desired DNA using PolyEthylenImine (PEI) as transfection reagent at the DNA:PEI ratio of 1:3. Expression plasmids for V 2 R, B 2 R, AT 1a R, Ib30-YFP, barr1 shRNA, and barr1-mCherry have been described previously (Kumari et al, 2016(Kumari et al, , 2017Ghosh et al, 2017Ghosh et al, , 2019Pandey et al, 2019a;Baidya et al, 2020). Receptor mutants were generated using site-directed mutagenesis kit (NEB) and sequenced (Macrogen) before use in the experiments.…”

“…A subsequent study however differentiated B 2 R from other class B GPCRs including V 2 R, by reporting that barrs rapidly dissociate from this receptor in endosomes followed by receptor recycling to the plasma membrane (Simaan et al, 2005). The most interesting aspect that prompted us to choose these two receptor systems is that previous studies have demonstrated strikingly different contribution of barr1 in agonistinduced ERK1/2 phosphorylation for these two receptors (Ren et al, 2005;Charest et al, 2007;Zimmerman et al, 2011;Oligny-Longpre et al, 2012;Ghosh et al, 2019). While barr1 knockdown leads to a significant reduction in ERK1/2 phosphorylation downstream of V 2 R (Ghosh et al, 2019), it results in an enhanced level of ERK1/2 phosphorylation for the B 2 R (Zimmerman et al, 2011).…”

“…In order to gain additional mechanistic insight into differential contribution of barr1 in ERK1/2 activation for V 2 R and B 2 R, we used a previously described intrabody-based sensor of barr1 conformation in cellular context (Ghosh et al, 2019;Baidya et al, 2020). This sensor is based on a synthetic antibody fragment referred to as Fab30 that selectively recognizes V 2 R-bound barr1 conformation in vitro (Shukla et al, 2013(Shukla et al, , 2014Kumari et al, 2016Kumari et al, , 2017Ghosh et al, 2019). Moreover, an intrabody version of Fab30, referred to as Ib30, also recognizes barr1 upon agonist stimulation of V 2 R in cellular context (Ghosh et al, 2019).…”

“…This sensor is based on a synthetic antibody fragment referred to as Fab30 that selectively recognizes V 2 R-bound barr1 conformation in vitro (Shukla et al, 2013(Shukla et al, , 2014Kumari et al, 2016Kumari et al, , 2017Ghosh et al, 2019). Moreover, an intrabody version of Fab30, referred to as Ib30, also recognizes barr1 upon agonist stimulation of V 2 R in cellular context (Ghosh et al, 2019). Recently, a YFP-tagged version of Ib30 has also been generated and used as a conformational probe for measuring the interaction and trafficking of barr1 in cells upon GPCR stimulation (Baidya et al, 2020).…”

I8-arachnotocin–an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor

“…Since recent studies uncovered an overlapping role of β-arrestin-1 and β-arrestin-2 with regard to V 2 R-dependent agonist-induced endocytosis and ERK activation 28 , we probed whether I8-arachnotocin is capable of recruiting β-arrestin-1 in a BRET-based assay. In comparison to VP (10 µM), which robustly induced β-arrestin-1 recruitment, I8-arachnotocin (10 µM) also failed to recruit β-arrestin-1 in a time-dependent manner (Fig.…”